HU186839B - Method dor determining the amount of igc aeing in new-born calf, foal or colostrum of cow or mare - Google Patents

Abstract

A method is provided for determining the amount of IgG present in a neonatal foal or calf or in the colostrum of a dam or cow. The method involves contacting a body fluid sample with biologically inert latex particles, measuring the amount of agglutination which occurs, and determining the amount of IgG present. The latex particles are diluted to a final concentration of from about 0.65 to 2.0 percent (w/v) at a pH of from about 7.5 to 9.0 with a buffer. The body fluid is diluted to a range of from .01:12 parts to .01:2160 parts, v/v basis, at a pH of from about 7.5 to 9.0.

Description

The present invention provides an expansive method for determining the amount of IgG in a newborn calf, foal or colostrum of a cow or mare.

Newborn humans or animals acquire immunoglobulins, or antibodies, through the placental septum before they are born. It is primarily in the presence of these immunoglobulins that the newborn develops an appropriate level (concentration) of antibodies that renders the newborn resistant to infectious diseases. One type of these immunoglobulins, IgG, occurs very early on under the influence of an immunogenic stimulus and, by distribution primarily through blood vessels, represents the first defense system.

In contrast, foals and calves have virtually no IgG until the placenta and therefore have only very low IgG levels at birth; this condition is called hypogammaglobulinemia. This situation usually changes with the uptake of IgG-rich colostrum and its absorption through the intestinal epithelium. Mammals should absorb IgG within approximately 24 hours after birth because after this time, IgG is no longer absorbed by the intestine.

Absorption of the IgG content of the colostrum is important for the health of foals and calves in their neonates. If enough IgG is not absorbed, this is one of the main factors that cause otherwise normal foals to become infected and die [J. Am. Vet. Med. Ass., 166, 71 (1975)].

If IgG is not transmitted, it should be quickly and accurately recognized (diagnosed) to determine whether a therapeutic solution is available or whether the animal should be abandoned. This therapy may consist of injecting IgG.

Several publications deal with the determination of immunoglobulin by "latex flocculation assay", which means the flocculation of polystyrene-latex particles in a fluffy form. In this assay, latex particles are generally coated with an antigenic material such as hormones or blood proteins such as albumin or immunoglobulin such as IgG and IgM. The latex particles are negatively charged and the protein is bound by adsorption. In the second step, a second reagent is added to the latex particles, which causes the coated latex particles to adhere (agglutinate).

The Aust. Vet. J. 56, (1980) describes an attempt to detect the adsorption of colostrum immunoglobulins in newborn foals. In this assay, the latex particles and antiserum are mixed with purified equine immunoglobulin to coat the latex particles with Ιό-IgG antibodies. After incubation, the uncoated horse IgG from the coated latex particles is then washed with fresh buffer and reconstituted with buffer. The antibody latex mixture is then added to a test sample taken from foal plasma. The appearance of lumpy white agglutination means that the assay for the presence of immunoglobulins is positive.

Am. J. Med., 21, 888 (1956) describes the use of a la2 tex flocculation assay for the serological diagnosis of rheumatoid arthritis. They use an IgG immunoglobulin called rheumatoid factor (RF). The latex particles are first coated with human gamma globulin, and after incubation, the non-adsorbed gamma globulin is then washed with fresh buffer solution and then reconstituted with buffer solution. The mixture of gamma-globulin and latex is then added to a serum sample of a rheumatoid arthritis patient, which results in agglutination of 71 boars of arthritis patients. If the assay is modified by mixing the suspension of latex particles and serum without gamma globulin, only 11% of rheumatic sera will agglutinate.

U.S. Patent No. 3,088,875 discloses a latex flocculation assay for the detection of a "C-reactive" protein, which occurs mainly in the serum of patients with active inflammation or tissue destruction. In this test, the latex particles are mixed with human gamma globulin and then heated to 57 ° C. The antibody coated latex particles are then mixed with diluted serum from a patient. The presence of agglutination proves the presence of C-reactive protein.

U.S. Patent 3,551,555 discloses an assay that can be used to detect an antigen, such as human chorionic gonadotropin. In this assay, the latex particles are first coated with an inert protein, such as albumin or lactalbumin, and then coated with either an antigen or an antibody. The protein antibody or protein antigen coated latex particles are then mixed with diluted serum from a patient. The onset of agglutination indicates the presence of an antigen.

According to all the patents and publications mentioned above, the latex particles are first coated with a proteinaceous material and then the latex particles so coated are interacted with a second reagent. Thus, the second reagent creates a crosslinking between the latex particles and "bridging" agglutination occurs.

No disclosure makes or suggests that IgG levels (concentrations) in foals or calves or levels (concentrations) of IgG in the colostrum of a mare or cow can be determined without the use of an antibody-antigen reaction.

The present invention relates to a method for determining the amount of IgG in a newborn foal or calf or in a colostrum of a mare or cow. The method of the present invention comprises contacting a sample of body fluid with biologically inert latex particles and determining the extent of agglutination to occur and thereby determining the amount of IgG present in the sample. The latex particles are diluted to a final concentration of about 0.65-2.0% w / v at pH 7.5-9.0 using a buffer solution. The body fluid is diluted at a pH of about 7.5 to about 9.0 with a buffer solution of about 0.01: 12 to about 0.01: 2160 v / v.

Critical levels (concentrations) of IgG are likely to be: below 200 mg / dl therapy required;

Between 200 and 400 mg / dL, the question of whether therapy is needed; and concentrations above 400 mg / dl are satisfactory. The required process makes it easy to measure these concentrations.

The latex particles used in the process of the invention may be any suitable biologically inert particles. Suitable particles are, for example, polyvinyl toluene, styrene-butadiene latex, styrene / divinylbenzene-latex, acrylic latex and polystyrene latex. The size of the latex particles may range from 0.109 to 0.81 µm. Such latex particles are available, for example, from the following companies; Dow Chemical Co. (Midland, Michigan, USA); Monsanto and Co. (St. Louis Missouri, USA); Rhone-Poulenc (Paris, France); and AB Bofors (Örebro, Sweden).

The latex particles are commercially available in the form of suspensions, for example 10% w / v aqueous latex suspension and can be diluted with a suitable buffer solution. Buffers which are sufficiently soluble in water and have a high buffer capacity at a pH of about 7.5 to about 9 are suitable. Suitable buffers are, for example, a mixture of borate and physiological saline, a mixture of glycine and physiological saline, and N, N-bis (2-hydroxyethyl / glycine).

Suitable borate buffer available for example by mixing HCI / ml and 7.1 mm 12.2 mmol Na2B from ₄ O7. To this borate solution is added 0.08-10% w / v saline. A 0.3 to 1.25 weight percent solution is preferred. If desired, the buffer consisting of borate and physiological saline may be prepared by 50 ml of 0.1 M boric acid and

5.9 ml of 0.1 N sodium hydroxide solution are added, the mixture is made up to 100 ml with water and adjusted to a pH of about 8.2, and 0.85 g of sodium chloride is then added per 100 ml of buffer solution.

A suitable buffer solution containing glycine and physiological saline is 0.1 M, pH 8.2, and contains 10 g of sodium chloride per liter. The latex particles are diluted to a final concentration of 0.65 to 2% w / v at a pH of about 7.5 to about 90.

The body fluid may be the plasma, blood or whole blood of a newborn foal or calf, or may be derived from the colostrum of a mare or cow. The body fluid is diluted with one of the aforementioned buffers so that the volume ratio of body fluid parts to buffer parts is 0.01: 12 to 0.01: 2160.

Experimental results have shown that limiting the particle size to a size range of 0.109-0.81 µm is essential. As the size of the latex particles decreases, the specific surface area increases significantly, which makes it difficult to detect agglutination. As the size of the latex particles increases, the specific surface area decreases significantly.

The same consideration applies to limiting the amount of latex particles to a concentration range of 0.65 to 2.0% by weight. If the concentration of latex particles is greater than 2.0% by weight, the determination of IgG cannot be performed because too much IgG is required. However, when the concentration of latex particles falls below 0.65% by weight, the detection of agglutination becomes increasingly difficult.

Using the above-mentioned latex sizes and concentrations, it has been determined that the body fluid to be tested can be diluted so that the volume ratio of body fluid to volume relative to buffer is 0.01: 12 to 0.01: 2160. Diluting the body fluid to a ratio of less than 0.01: 2160 relative to the buffer would require particles larger than desired to cause agglutination within the same concentration of IgG and would therefore be difficult to operate with such a system. If the body fluid is more concentrated at a ratio of 0.01: 12 relative to the buffer solution, agglutination will occur in the presence of insignificant IgGcc concentrations in addition to all the aforementioned sizes of latex particles.

The correct dilution of latex and serum with an appropriate buffer solution can be readily determined by one of ordinary skill in the art, as described below. In a preferred embodiment of the invention, the latex particles having a size of 0.22 µm are diluted to a final concentration of 2.0% by weight.

The process of the invention is illustrated in detail in the following embodiments, unless otherwise noted, latex solutions were obtained from Dow Chemica: Co.

Process control

Serum samples were taken from newborn foals approximately 6-8 hours after birth. The Radial Immunodiffusion Assay Kit ("RID" (purchased from Miles Laboratories Inc., Elkhart, Indiana, USA)) was used as follows: The kit contained Ιό-IgG antibody (antibody) embedded in buffered agarose. The standard amount of serum to be measured was filled into one well, and the standard control samples were placed in the other wells. Serum and agarose were incubated at room temperature for about 16-24 hours. A precipitate was formed around each well proportional to the amount of IgG present The diameter of these rings was measured and plotted against semi-logarithmic paper as a function of concentration, and the amount of IgG in the serum samples was determined on this basis.

Example 1 A polystyrene latex solution containing 0.22 pm by weight / volume of a 0.2 M solution of N, N-bis (2-hydroxy) in a ratio of 1: 5 by volume to 8.5. ethyl glycine was diluted to a final concentration of about 2% w / v.

Foal serum samples were used in duplicate for each assay, as in the control procedure. In a series of assays, it was found that IgG concentrations can be readily determined if the required serum volume is no greater than 4-6 drops. Based on these experiments, 5 µl of foal serum was added to 8 ml of N, N-bis (2-hydroxyethyl) glycine buffer solution (0.2 M, pH 8.5), and

-3186839 was thoroughly mixed (0.01 volume of sample to 16 volumes of buffer).

Each drop of the diluted latex mixture contained 0.025 mL; the diluted latex droplet contained 0.10 ml. Two drops of diluted latex mixture were placed on a slide, one drop of diluted foal serum diluted as described above, and mixed. The slide was gently rotated and observed for agglutination in the mixture. If after 10 seconds no visible agglutination was observed, an additional 1 drop of diluted serum was added and mixed as above. This titration was continued until agglutination occurred. This procedure was repeated for each diluted foal serum sample 1.

The results of the IgG agglutination assays thus obtained were compared with those of the control IgG assay. The test results are summarized in Table 1. ⁵

Out of a total of 77 samples tested, 73 (94.8%) were consistent with the IgG concentrations obtained during the RID control procedure. The same results were obtained even if the accuracy of these 5 assays was determined by Eq. Vet. 6. 109 (1974) compared with the zinc sulfate assay.

These test results show that by using the method of the invention, the IgG concentration of ⁰ concentrations are compared with a recognized, practical tests carried out can be determined with high precision.

The specific reaction conditions used - i.e., when the size of the latex particles is 0.22 µm and the concentration of la ⁵ tex solution is 2.0% w / v and the ratio of volume of body fluid to volume of buffer solution are 0.01: 16 - IgG concentration can be determined in a simple manner using 4-5 drops of diluted body fluid.

Table 1

IgG concentrations obtained by RID measurement Test method - amount of serum added > 400 mg / dL 1 drop of diluted serum 2 drops for diluted latex; agglutination is positive £ 400 mg / dL 2 drops; agglutination is positive 200-400 mg / dL 3 drops: Agglutination is positive Ξ200 mg / dL 4 drops: Agglutination is positive <200 mg / dL > 4 drops: Agglutination is positive

Using the criteria in Table 1, a series of 77 foal serum samples were tested according to the method of the invention and compared to the IgG concentrations determined during the RID control procedure. Our results are summarized in Table 2.

Example 2

Serum samples were taken from two foals and RID assay showed IgG concentrations of about 1500 mg / dL and about 0-200 mg / dL, respectively. Samples were pooled to give a series of IgG concentrations (mg / dL) of 750,

500, 373, 300, 250, and 166. These serum samples were used as follows.

a polystyrene latex having a particle size of 0.22 μτη w / v, mixed with 0.2 M N, N-bis (2-hydroxyethyl) glycine buffer, pH 8.5, at a ratio of about 2 to about 2 vol. diluted to a final concentration by weight / volume.

For each assay, 5 μΐ of Foal Serum at the above concentrations was added to 6 mL of the above buffer solution and adjusted to a final concentration of _{n by} mixing with 12 volumes of 0.01 volumes of buffer. 1 drop of diluted latex solution on the slide was mixed with 1 drop of diluted serum, vortexed and agglutination observed. If no agglutination occurs, 1 additional diluted latex sample is added to the slide 2 drops. spreadsheet

IgG Group Percentage (agglutination) of the samples relative to the assay

AUiívcuu awv (RID) mg / dl wania a group * 1 1.5 * 2 * 2.5 * * 3 3.5 * * 4 4.5 * * 5 5.5 * > 400 52 (8) (12) (28) (4) 0 0 0 0 0 0 15.3% 23% 53% 7.7% 200-400 16 0 0 0 (2) (9) (3) (1) (1) 0 0 12.5% 56.3% 18.3% 6.25% 6.25% 200 9 0 0 0 0 0 0 (6) (1) 0 (2) 60% 10% 20% altogether 77 samples

* The number of drops was mixed with serum. If no agglutinator was present, the procedure was repeated with 3 drops of diluted serum. The test results are a

Table 3 shows

Table 3

The amount of serum added

IgC mg / di (by RID measurement)

750x2 = 1500 1 drop 500x2 = 1000 1 drop 373x2 = 750 ΐ drop 300 X 2 = 600 1 drop 250x2 = 500 1 drop 2 drops

agglutination positive is agglutination positive is. agglutination positive agglutination positive agglutination negative agglutination positive

166x2 = 332 and 2 drops: agglutination negative 3 drops: agglutination positive

The test results shown in Table 3 were compared with the titration results obtained in Example 1. Since the titration results, as shown in Table 1, were obtained by adding 1 drop of serum to 2 drops of diluted latex, the concentrations shown in Table 3 were multiplied by 2 to make Table 1 comparable to Table 3.

The results show that the IgG concentrations obtained in Example 2 are closely related to the IgG concentrations obtained in Example 1 titration (which are related to the RID control procedure). For example, at an IgG concentration of 66 mg / dL (166 x 2 = 332 mg / dL), 3 drops of serum gave positive agitation; it is in the range of 200-400 mg / dl, as shown in Table 1 by agglutination with 3 drops. Similar results were obtained for the other IgG concentrations.

It will be appreciated that by performing the procedure described in Example 1 and Example 2, the agglutinator of body fluid and Satex, and hence the concentration of IgG present in the samples, can be determined. One skilled in the art can adjust the desired concentration of the buffer solution and the size and concentration of the latex particles to determine the concentration of IgG in a body fluid. These concentrations can be easily optimized to provide a convenient IgG assay.

In the following examples, the reaction conditions are varied within wide limits. These reaction conditions are not necessarily the most favorable (optimal): as a result, the resulting agglutination values, i.e., IgG concentrations, in many examples are not directly comparable to those obtained in Example 1. However, in each example, to the extent that the amount of IgG present in the sample was increased or decreased, the agglutination changed so that the IgG was measurable by the method of the invention.

Unless otherwise noted, concentrations of foal sera in the examples below were obtained as in Example 2.

In order to determine the effect of modifying the particle size to 0.10 / ounce, the following experiment was performed.

Example 3

A 10% aqueous solution of polystyrene-butadiene la-alpha of 0.109 ptn was adjusted to a final concentration of 0.67% w / v by dilution 5: 15 in the buffer described in Example I.

In each of our assays, 5 µl of foal serum was added to 6 ml of the above buffer solution so that it was thoroughly reconstituted to a final concentration of ¹ 12 volumes of buffer containing 0.01 volumes of serum. 1 drop of the diluted latex mixture is shown in FIG. Example 1 was mixed with 1, 2 or 3 drops of dilute-stem n. The experimental results obtained are shown in Table 4.

Table 4

Ugg mg / dl (RID measurements)

750x2 = 1500

500x2 = 1000 25 375x2 = 750

300x2 = 600

Amount of serum added drop: agglutination negative drop: agglutination positive drop: agglutination negative drop: agglutination positive drop and 2 drops: agglutination negative drop: agglutination positive 1, 2 and 3 drops: agglutination negative

It follows from the above data that as the amount of IgG present in the serum samples decreased, the amount of serum required for agglutination increased. The reduction in the size of the latex particles has led to an adequate female kidney of the specific surface and consequently a reduction in the concentration of the latex particles has become necessary.

In Examples 4 and 5 below, stripe serum samples were diluted to obtain a series of IgG concentrations shown in the respective tables.

Example 4

Részecskenréretű 0.497 pm polystyrene latex aqueous solution of 10% 1 buffer of Example 1 to about 2 wt / vol .-% final concentration was adjusted by dilution to 5 ⁴⁵ Ban ratio.

In each of our assays, 5 μó of foal serum was added to 108 ml of the above buffer solution and the mix was thoroughly mixed to a final concentration of 216 volumes of buffer containing 0.01 volumes of serum. 1 drop of diluted latex was mixed with 1, 2 or 3 drops of diluted serum as described in Example 1. The experimental results thus obtained are as follows

Table 5 contains.

Table 5

Mg of mg / dl (Rll) ',) ⁶⁰ 900x2 = 1800 370x2 = 740

200x2 = 400 65 ______, ___

Amount of serum added drop: agglutination positive drop: agglutination questionable drop: agglutination positive drop: agglutination negative drop: agglutination positive

The above data show that as the amount of IgG used in the examples decreased, the amount of serum sample required for agglutination increased. The dilution of the serum sample had to be increased because increasing the size of the latex particles resulted in a decrease in the specific surface area of the latex particles.

Example 5

A 10% aqueous solution of polystyrene latex having a particle size of 0.807 µm was adjusted to a final concentration of about 2% w / v by dilution 1: 5 with the buffer described in Example 1.

In each assay, 5 µl of foal serum was added to 1080 ml of the above buffer solution and adjusted to a final concentration by thorough mixing so that 2160 volumes of buffer solution contained 0.01 volumes of serum. 1 drop of diluted latex was mixed with 1-3 drops of diluted serum on the slide as described in Example 1. The experimental results obtained are shown in Table 6.

Table 6

IgG mg / dl (by RID measurement)

900 x 2 = 1800 370x2 = 740

200 x 2 = 400

Serum added drop: agglutination positive 1 drop: agglutination negative 3 drop: agglutination positive 3 drop: agglutination negative

It follows from the above data that as the amount of IgG present in the serum samples decreased, the amount of serum sample required for agglutination increased. The above data also shows the effect that is evident from Example 4: an increase in the size of the latex particles leads to a decrease in the specific surface area of the latex particles and consequently a further increase in the dilution of the serum sample is required.

Example 6

Add 10 g of sodium chloride to 1 liter of the buffer solution of Example 1 to give a N, N-bis (2-hydroxyethyl) glycine buffer solution containing physiological saline. A 10% aqueous solution of polystyrene butadiene latex having a particle size of 0.109 µm was diluted 1: 5 with this buffer solution to a final concentration of about 0.67% w / v.

In each assay, 5 µl of foal serum was added to 6 ml of the above solution and mixed thoroughly. 1 drop of diluted latex solution was mixed with 1, 2 or 3 drops of diluted serum on the slide as described in Example I. The experimental results obtained are shown in Table 7.

Table 7

IgG mg / dl (by RID measurement) ^J 750X2 = 1500 500x2 = 1000 375x2 = 750 300x2 = 600 _Q 250x2 = 500 166x2 = 332

Amount of serum added drop: agglutination positive 1 drop: agglutination positive 1 drop: agglutination positive 1 drop: agglutination positive.

drop: agglutination negative drop: agglutination positive and 2 drops: agglutination negative drop: agglutination positive ¹⁵ It follows from the above data that as the amount of IgG in the serum samples decreased, the amount of serum sample required for agglutination increased. In addition, the results also show what can be achieved by selecting the buffer ^20. The addition of saline to the buffer solution results in the 109 µm latex particles behaving more like the 0.22 µm latex particles of Example 2.

²⁵ Foal serum samples 3000 mg / dl and 1500 mg / d! Mixed with IgG into a series, assayed by RID and used in the following example.

Example 7

A 10% aqueous solution of polystyrene latex having a particle size of 0.22 μπι was adjusted to a final concentration of about 2% w / v by dilution with 0.1 M glycine buffer pH 8.2.

In each assay, 5 µl of foal serum was added to 6 ml of the above glycine buffer and mixed thoroughly. 1 drop of diluted latex was mixed with 1, 2 or 3 drops of diluted serum as described in Example 1 on the slide. The experimental results obtained are shown in Table 8.

Table 8

IgG mg / dl (by RID measurement) The amount of serum added 3000X2 = 6000, _n 1500x2 = 3000 bu Drops 1 and 2: agglutination negative 3 drops: agglutination positive Drops 1, 2 and 3: Agglutination negative

Again, the decrease in the amount of IgG in the serum sample results in an increase in the amount of serum sample required for agglutination. The presence of glycine in the buffer solution, instead of the buffer used in the previous example, requires a higher amount of IgG for agglutination.

⁶⁰ Example 8

A 10% aqueous solution of polystyrene latex having a particle size of 0.22 μπι, diluted 1: 5 with 0.1 molar buffer solution of glycine and physiological saline solution, pH 8.2, containing 10 g of sodium chloride per liter. .-% final concentration.

In each assay, 5 mL of foal serum was added to 6 mL of glycine saline buffer and mixed well. 1 drop of diluted latex on the slide

As described in Example 1, 1, 2 or 3 drops of diluted serum were mixed. The experimental results obtained are shown in Table 9.

Table 9

The amount of serum added

IgG mg / dl (by RID measurement)

750x2 = 1500 1 drop 500 x 2 = 1000 1 drop 375x2 = 750 1 drop 300 x 2 = 600 1 drop 250x2 = 500 1 drop 2 drops 166x2 = 332 1 and 2 tsp 3 drops

agglutination positive agglutination positive agglutination positive agglutination positive agglutination negative agglutination positive pp: agglutination negative agglutination positive

Again, the decrease in the amount of IgG present in the serum sample requires an increase in the amount of serum sample required for agglutination. Addition of saline to the glycine buffer solution of Example 7 significantly increases the sensitivity of the IgG assay. While glycine alone, when used as a buffer, requires a high amount of IgG for agglutination, the sensitivity of the buffer consisting of a mixture of glycine and physiological saline is similar to that of the N, N-bis (2-hydroxyethyl) glycine buffer solution of Example 2. .

Example 9

A 10% aqueous solution of polystyrene latex having a particle size of 0.22 µm, diluted 1: 5 with a buffer solution of borate and physiological saline solution (0.125 mol, pH 8.4, 8.5 g / l) by volume to a final concentration.

This buffer solution was prepared by making a mixture of 50 ml of 0.1 M boric acid solution and 5 ml of 0.1 N sodium hydroxide solution to 100 ml with water, adjusting to pH 8.2, mixing and adding 8.85 per 100 ml of buffer solution. g of sodium chloride was added.

In each assay, 5 ml of foal serum was added to 6 ml of borate and saline buffer and mixed thoroughly. A drop of diluted latex was mixed with 1 or 3 drops of diluted serum on a slide as described in Example 1. Our assay results were essentially the same as those obtained using glycine saline buffer as described in Example 8.

Example 10

A 10% aqueous solution of polystyrene-butadiene latex having a particle size of 0.109 µm was diluted 1: 5 with the buffer solution of Example 1 to a final concentration of about 2%. Foal serum samples described in Example 1 were prepared for each of our assays. 5 ml of foal serum was added to 10 ml of the above buffer solution and mixed thoroughly. 1 drop of the diluted serum is as described in Example 1, 1 drop

It was added to 5 diluted latex. The experimental results obtained are shown in Table 10. Table 70 10 IgG mg / dl (by RID measurement) The amount of serum added 15 900 x 2 = 1800 370x2 = 740 200 x 2 = 400 4 drops: Agglutination is positive 7 drops: Agglutination is positive 9 drops: Agglutination is positive

The addition of an additional 1 drop of diluted latex can reverse the agglutination and, if further diluted serum is added, agglutination again occurs. This reversibility of agglutination shows that the endpoint can be determined with complete accuracy.

Example 11

The procedure of Example 1 and Example 2 was repeated, but using newborn calf serum instead of foal serum. The test results showed that as the amount of IgG present in the serum samples decreased, the amount of serum samples required for agglutination increased. The test results further confirmed that the method of the invention is suitable for measuring agglutination, i.e., for determining the amount of IgG in calf serum.

Example 12

The procedure described in Example 1 was repeated, however, using whole blood of foal and whole calf blood instead of foal serum. The test results showed that the method of the invention is suitable for measuring agglutination, i.e., for determining the amount of IgG present in whole blood of foals and calves.

⁴⁵

Example 73

The procedure of Example 1 was followed, however, using colostrum instead of serum to determine if the colostrum of a mare or cow that had just been shelled or calved was able to transfer the immunoglobulin to a newborn foal or calf.

A suitable colostrum IgG concentration is approximately 1000 mg / dL or greater. In order to dilute the colostrum sample to a concentration range approximating the serum concentration of Example 1, the colostrum was diluted 1: 5 with an appropriate buffer solution to a final concentration of about 200 mg / dL.

0.22 µm 10% w / v polystyrene solution diluted 1: 5 by volume with 0.2 M N, N-bis (2-hydroxyethyl) glycine buffer, pH 8.5, approx. It was set to a final concentration of 2% w / v.

-7 '

In each assay, 5 µl of diluted colostrum was added to 8 ml of the above buffer solution and diluted to a final concentration such that 16 volumes of buffer contained 0.01 volumes of colostrum. This solution was used as in Example 1 for titration of 2 drops of latex.

Claims (5)

1. A method for determining the amount of IgG in a neonatal foal, calf or colostrum of a mare or cow, comprising contacting a sample of the neonatal calf or foal body fluid or colostrum with biologically inert latex particles, and reducing the degree of agglutination present. is determined, during which time 0.109-0.81 pm. size latex particles are diluted to a final concentration of about 0.65-2.0% w / v with a buffer solution at a pH of about 7.5 to about 9.0 and a sample of body fluid or colostrum is diluted with a buffer solution at a pH of about 7.5 to about 9.0. is diluted such that the volume fraction of sample relative to volume of buffer solution 5 ranges from 0.01: 12 to 0.01: 2160. 2. A method according to claim 1 wherein the body fluid is blood plasma, blood serum or whole blood. 3. The method of claim ¹⁰ the first embodiment mode, characterized in that a body fluid is colostrum. Fourth embodiment of the method according to claim 1, characterized in that it is used borate and saline mixtures, or glycine, and saline solution ¹⁵ was, or N, N-bis (2-hydroxyethyl) glycine and saline mixture as a buffer. 5. The process of claim 1 wherein 0.22 µm of latex particles are used and the ratio of body fluid to puf ³⁰ is 0.01: 16. Without illustration